Influenza viruses rapidly accumulate mutations in antibody (Ab) binding sites within the hemagglutinin (HA) and neuraminidase (NA) proteins, a process termed 'antigenic drift'. Due to antigenic drift, humans are typically re-infected with distinct influenza strains. Although most humans are sequentially infected with different influenza strains over the course of their life, annual vaccine strains are chosen based on serological studies utilizing anti-sera prepared in ferrets recovering from a primary influenza infection. Accurate selections of vaccine strains are therefore based on the assumption that reference sera created in ferrets are representative of immunity found in the human population. Our preliminary data strongly suggest that influenza Ab specificities in humans are strongly influenced by previous influenza infections. This proposal will test the hypothesis that human influenza viruses evolve in response to Ab repertoires elicited by sequential influenza exposures, and that anti-sera prepared in ferrets fail to detect genuine antigenic changes that prevent binding of human Abs. In AIM 1 we will determine if recent pandemic H1N1 viruses have evolved to prevent binding of Abs elicited by sequential H1N1 exposures. For these studies, we will test if sera isolated from sequentially exposed ferrets and humans bind to pandemic H1N1 viruses engineered to possess new HA mutations that are now present in most circulating pandemic H1N1 strains. In AIM 2, we will determine how pre-exposure history influences Ab responses against H3N2 viruses. H3N2 viruses have circulated in the human population for 45 years, and the genetic and antigenic evolution of these viruses is well studied. We will sequentially infect mice and ferrets with different H3N2 strains and determine if Abs elicited in these animals recognize H3N2 viruses engineered to possess mutations that have arisen in distinct antigenic sites over the last 45 years. Finally, in AIM 3 we will determine how different pre-exposures affect vaccine responsiveness in infants. We will measure Ab specificities in sera from infants receiving 2 influenza vaccines from the same season (prime- boost with same antigen) and infants receiving 2 antigenically distinct influenza vaccines from different seasons (prime-boost with different antigenically distinct antigens). Together, these studies will further our understanding of the immunological pressures that promote antigenic drift of influenza viruses. These studies will shed light on why influenza vaccines elicit ineffectve responses in different individuals and will also improve the process by which vaccine strains are chosen.